Effect of contrasting phosphorus levels on nitrous oxide and carbon dioxide emissions from temperate grassland soils

Agricultural practices such as repeated fertilization impact carbon (C), nitrogen (N) and phosphorus (P) cycling and their relationships in the plant–soil continuum, which could have important implications for the magnitude of greenhouse gas emissions. However, little is known about the effect of C and N additions under contrasting soil P availability status on nitrous oxide (N2O) and carbon dioxide (CO2) emissions. In this study, we conducted a field-based experiment that investigated the impact of long-term (23 years) P management (no (P0, 0 kg P ha−1), low (P15, 15 kg P ha−1) and high (P45, 45 kg P ha−1) P inputs) on N2O and CO2 emissions following two C + N application events in two managed grassland ecosystems with loam and sandy loam soils. The magnitude of fluxes varied between the soil P availability levels. Cumulative N2O emission was significantly higher in P0 soils (1.08 ± 0.09 g N2O-N m−2) than P45 soils (0.63 ± 0.03 g N2O-N m−2), with the loam soil (1.04 ± 0.04 g N2O-N m−2) producing significantly higher emissions than the sandy loam soil (0.88 ± 0.05 g N2O-N m−2). We conclude that P-limitation stimulates N2O emissions, whereas P-enrichment promotes soil respiration in these temperate grassland sites. Our findings inform effective nutrient management strategies underpinning optimized use of N and P inputs to agricultural soils as mitigation measures for both food security and reducing greenhouse gas emissions.

www.nature.com/scientificreports/ nutrients and carbon [16][17][18] . Nitrous oxide is primarily produced through microbial nitrification and denitrification, and its production via these pathways are affected by microbial composition as well as the availability of soil mineral N and phosphate, C substrate, oxygen, soil moisture, pH, and soil temperature 17,19 . While it is known that N fertilizer applications contribute to the formation of N 2 O 4 , there is a poor understanding of the interaction between soil nutrients and carbon availability, and their subsequent impact on N 2 O and CO 2 emissions in agricultural soils. Fertilizer-driven changes in managed grassland soils could have functional implications for the composition of arbuscular mycorrhizal fungi (AMF) and alter their symbiotic relationship with plants 15,[20][21][22] . This symbiotic relationship comprises of increased access to N and P facilitated by the AMF to the plant in return for C 11,23 . However, the extent to which N, P and C is exchanged could be influenced by an increasing use of N and P fertilizers. In a P-rich soil, N enrichment has been found to cause reduced allocation of photosynthates to mycorrhizae arbuscules, coils and extraradical hyphae 23 . In contrast, nitrogen enrichment of low P soils increased C allocations to these structures 23 . Thus, the availability of N relative to P in mycorrhizal system could affect the relationship between fungi and hosting plants and limit the ability of fungi to procure the elements. Lower P levels were associated with a significant increase of AMF colonization in a study conducted in the same experimental field of the current investigation 24 . Contrasting presence of AMF in agricultural soils could have major implications for variable N 2 O and CO 2 formation and may result in different nutrient use of efficiency of plants. Bender et al. 25 and Storer et al. 26 showed reduced N 2 O emissions in soils with abundant presence of fungi group, AMF, despite fungi are generally considered as a source of N 2 O as they lack N2O reductase 27 . In contrast to these findings, Okiobe et al. 28 demonstrated promoted potential N 2 O production as a result of decreased abundance of arbuscular mycorrhizal fungi. In a recent laboratory-based study, significantly higher N 2 O emission was observed in a P-limited soil than in a P-enriched soil following the same input of C and N in the two varying P-levels 29 . However, this relationship requires further investigation and verification under natural field conditions with plants present. Here we investigate the influence of N fertilizer addition and C availability on N 2 O and CO 2 emissions across two agricultural soils with sandy loam (Site A) and loam (Site B) textures with differing soil P levels in each soil 30 . We hypothesized that the largest magnitude of N 2 O release occurs at low soil P levels in grassland soils. We further hypothesized that CO 2 production increases with increasing levels of P in these soils. The main objective was to understand how N 2 O and CO 2 emissions are affected in response to C + N additions across a soil P gradient.

Materials and method
Site description. This experiment was conducted in two long-term P-trial grassland sites (Site A and Site B) situated in proximity (~ 350 m) to each other in the dairy farm at Johnstown Castle, Wexford, Co. Wexford, Ireland (6°49′ W, 52°29′ N). The sites were grazed permanent grasslands before establishment. When the experiment was established in 1995, 16 (10 m × 2 m) plots were formed in each site in a fully randomised block design with four replicates. The two sites established were selected to represent different soil types and drainage classes. Site A is a moderately drained brown earth and site B is an imperfectly drained gley soil 31 . Each year in February, each plot received one of the four phosphorous (P) fertilization rates (16% P superphosphate): 0 (P0), 15 (P15), 30 (P30), and 45 (P45) kg P ha −1 year −1 . All plots were initially sown with Lolium perenne and reseeded in 2016 with the same species. However, plant species such as Poa trivialis, Agropyron repens, Trifolium repens were present to a lesser extent. Above-ground biomass is harvested each month between February and August followed by 40 kg N ha −1 fertilizer applications. In the year (2019) of this experiment and the years before, SulCAN as a solid was applied at the first or second week of each month during February-August and potassium (K) as muriate of potash (KCl) was applied in February at a rate of 125 kg K ha −1 . SulCAN contains 26.7% N in the form of nitric and ammoniacal nitrogen and 5% water soluble Sulphur. For this study plots receiving P0, P15 and P45 at the two field sites were set up to carry out this experiment. The two sites were selected as they had slightly different soil properties and thus there was an opportunity to consider a soil × treatment effect in the experiment. Experimental design. Fertilizer N and substrate C were applied on 8 May and 12 June in the experiment undertaken between May and July 2019, which represents the main growing season in Ireland. Within each plot, an area of 1 m × 1 m was selected. Following N fertilizer application (40 kg N ha −1 ) to all plots, carbon substrate [mixture of glucose (40%), sodium acetate (30%) and methanol (30%)] was applied once within the selected area using a sprayer watering can. Labile C available in animal excreta usually contains carbohydrates, volatile fatty acids, and alcohols 32 ; as such different carbon substrates were applied to mimic this. Our review of the literature also indicated that C source types could differentially affect denitrifying communities and consequently denitrification rate. Thus, a mixture of three C sources was used to decrease bias of one microbial group over another as a result of single substrate use. Carbon was supplied to alleviate C-limitations of denitrification and nitrification processes as observed by O'Neill et al. 29 in soils from this trial and to ensure equal substrate availability across all soil P levels. Equivalent C input rate of 0.63 g C m −2 day −1 was added to represent a daily rate of plant carbon input from Lolium perenne dominated ecosystem 33 . Soil samples were collected on eight occasions throughout the experimental period. Soil was sampled from across each selected area to a depth of 10 cm, sieved through 4 mm sieve and analysed for soil mineral N and microbial biomass.
Soil properties, plant biomass and climate parameters. Physico-chemical soil properties were characterized by taking samples from 10 cm depth from each plot in the two sites before the commencement of the experiment. Soil pH was measured in water (2:1, water volume:soil mass) using Sally pH Auto analyser Dilution System (Gilson 215, Gilson, Dunstable, England). Soil organic matter (SOM) content was determined from mass loss on ignition at 550 °C for 7 h. Total C and total N concentrations were measured using a TrueSpec C/N www.nature.com/scientificreports/ analyser (TruSpec, LECO Corporation, Michigan, USA). Plant available P, potassium (K), and magnesium (Mg) were estimated using Morgan's extraction 34 and analysed using a Lachat QuickChem 8500 Series 2 Flow injection Analyzer (Lachat, QuickChem, 5600 Loveland, Colorado, USA). Particle size analysis was performed using the Pipette method 35 , where 2 mm sieved dry soil (20 g) was pre-treated with 6% H 2 O 2 , 3% NH 4 OH, and 5% sodium hexametaphosphate before separating soil aliquots into particle sizes. Water Holding Capacity (WHC) was determined from the mass difference between water-saturated and then overnight dried (105 °C) soil. Bulk density was determined by dividing weight of oven-dried soil by the total soil volume. To determine the mineral N concentrations, ten gram fresh soil was extracted with 50 mL 2 M KCl (5:1 solution to soil ratio). The supernatant was filtered through Whatman No. 1 filter paper and filtrates were stored in a cold room at 4 °C for about a week until analysis. Ammonium (NH 4 + ) and nitrate (NO 3 − ) concentrations in the extracts were analysed by the Aquakem 600 discrete analyser.
Above-ground plant biomass from each plot of both sites was harvested twice during the experiment period (June 10 and July 11, 2019) to a height of ~ 5 cm using a Haldrup plot harvester. The total harvested biomass weight from each plot was recorded and a 100 g sub-sample was taken for dry matter (DM) analysis. Each fresh herbage sub-sample was weighed and placed in an oven at 70 °C for 3 days, and dry weight of the biomass was determined after re-weighing.
Rainfall records for the experiment period were obtained from a Met Éireann weather observing station located in Teagasc dairy farm in Johnstown Castle, Co. Wexford., situated within a 100 m distance from the experimental sites. Volumetric soil moisture content and temperature was measured to 5 cm depth on individual plots on each gas sampling occasion using a handheld theta probe (WET-2 WET Sensor, Delta-T Devices, Cambridge, England). Water-filled pore space (WFPS) were calculated from the soil moisture values, bulk density of the soils, and soil particle density (2.65 g cm −3 ).
Microbial biomass, glomalin-related soil protein and potential denitrification activity. Soils were analysed for microbial biomass nitrogen (MBN), phosphorus (MBP) and carbon (MBC) using the fumigation extraction method as described respectively in (Brooks et al. 36,37 , and Vance et al. 38 ). Five gram fumigated (24 h) and non-fumigated soil samples were extracted with 100 mL 0.5 M NaHCO 3 and analysed for P colorimetrically using an Aquakem 600 discrete analyser (Thermo Electron OY, Vantaa, Finland). In order to avoid the spike readings by the instrument due to the effervescent nature of NaHCO 3 , one millilitre of 10% HCl was added to 10 mL extracts and diluted to 50 mL using distilled water. Microbial P was calculated by subtracting the P concentration of non-fumigated samples from fumigated samples, and dividing the result by an extraction factor of 0.4 37 .
Microbial biomass C and N were determined similarly using chloroform fumigation method with extraction period of 48 h with 0.5 M K 2 SO 4 38 . The extracts of the fumigated and non-fumigated samples were analysed for total C and N using a TOC-L CPH/CPN analyser (Shimadzu, Tokyo, Japan), and the differences, divided by correction factors of 0.45 and 0.54, were used to estimate the microbial biomass C and N, respectively.
Glomalin is a glycoprotein produced by AMF and can be used as an indicator of mycorrhizal colonization in the plant root-soil interface 39 . Total glomalin-related soil protein (GRPS) was extracted by 90 min of autoclaving (121 °C) of 1 g air-dried soil in 8 mL of 50 mM sodium citrate adjusted to pH 8.0 with HCl 40 . Three additional sequential extractions were performed with the sodium citrate solution by autoclaving for 60 min until no redbrown color was visible in the last supernatant. After autoclaving, the samples were centrifuged at 10,000 revolutions per minute (rpm) for 5 min. The amounts of glomalin in the extracts were quantified using the Bradford dye-binding assay with bovine serum albumin (BSA) as the standard (2 mg mL −1 ). In a 96-well plate, replicated 200 µL of standard or extracts and 50 µL of dye reagent were added in each well and mixed using a microplate mixer. The Bradford-reactive substance was determined by measuring absorbance at 600 nm using Microplate Reader (Modulus Microplate Multimode Reader, Turner BioSystems, Sunnyvale, California, USA). Sample concentrations were determined using the standard curve. Potential denitrification activity (PDA) was determined using the acetylene inhibition method, modified from Pell et al. 41 . Briefly, replicated 20 g fresh soils were added into two identical flasks from a sample of soil. The flasks were then sealed with a rubber stopper and flushed and filled with helium after evacuating the headspace air. In one of the replicas, 10% of the headspaces were removed and replaced by acetylene. All flasks were incubated at 15 °C on an orbital shaker at 175 rpm for 30 min followed by the addition of a nutrient solution containing 75 mmol L −1 KNO 3 , 37.5 mmol L −1 Na-succinate, 25 mmol L −1 glucose, and 75 mM Na-acetate. Gas samples were taken from the headspace every 1 h for 5 h. N 2 O concentrations were determined using a gas chromatograph (Bruker, Scion 456-GC, Livingston, Scotland), and PDA was calculated from the rate of change of N 2 O concentrations over time from acetylene amended flasks. N 2 O and CO 2 flux measurements. Gas samples (N 2 O and CO 2 fluxes) were measured before and after the application of N fertilizer and C substrates, with a daily sampling for 10 days directly after C + N additions and 3-4 times a week in the third and fourth week and 2-3 times a week in the subsequent weeks. A rectangular (40 × 40 cm) static collar, made of stainless steel (opaque), was anchored 5 cm deep into the soil within the marked area of 1 m × 1 m in each of the selected plots. During gas sampling, a 10 cm tall chamber lid fitted with two septa on top was placed on the collar lined with neoprene rubber band. To ensure hermetic sealing of the headspace during sampling, the ring area of the collar was half-filled with water, and a 10 kg weight was placed on the top of the lid to compress the seal. Gas samples were collected between 09:30 and 11:30 local time using a 10 mL Luer lock syringe fitted with a hypodermic needle via one of the septa at 0, 20, and 40 min after chamber closure. Prior to transferring the final sample into a pre-evacuated 7 mL glass vial, air in the chamber headspace was mixed by flushing the syringe three times. Gas samples were analysed using a gas chromatograph (Bruker, Scion 456-GC, Livingston, Scotland) fitted with an electron capture detector to analyse for N 2  where ∆C is the change in gas concentration in the chamber headspace during chamber enclosure period in ppbv, ∆t is chamber closing period in minutes, so ∆C/∆t is the slope of the gas concentration with time. M is the molar mass of N 2 O-N (28 g mol −1 ) and CO 2 -C (12 g mol −1 ), P and T are the atmospheric pressure (Pa) and temperature (K). Atmospheric pressure values were obtained from the nearby weather station whereas for T, wet sensor values were used. V is the headspace volume of the closed chamber (m 3 ) and A is surface are of the chamber (m 3 ). R is the ideal gas constant (8.314 J K −1 mol −1 ). Daily flux for each treatment is reported as the average of the replicates. Cumulative N 2 O and CO 2 emissions were calculated over each application period by multiplying the daily N 2 O and CO 2 fluxes by the number of days between two consecutive measurements. A summation of the cumulative flux of each application period is reported as the total cumulative flux.
Statistical analysis. ANOVAs with repeated measures were used to test for the C + N addition effect on N 2 O and CO 2 emissions, MBC, MBN, MBP, NO 3 − , and NH 4 + with P treatment, site, and day of measurement as fixed effects, and individual plots as a random effect. Two-way ANOVA was applied to test for main and interaction effects of P treatment and site on cumulative N 2 O and CO 2 emissions, soil property parameters (Table 1), plant biomass, and GRSP. Prior to analysis, response variables were checked for normality (sphericity for repeated ANOVA) and homogeneity of variance, and log transformed when required. Tukey's HSD post-hoc tests were conducted to identify pair-wise comparisons of significant effects at P < 0.05. We performed Spearman's rank correlation to assess the correlations between soil biophysicochemical parameters, plant biomass, and N 2 O and CO 2 emissions.
ANOVA analysis was performed using lmer function in lme4 package 42 within R software. All statistical analyses were conducted using R, version 3.6.0 43 .

Results
Soil properties and mineral nitrogen. Total C, total N, Mg, and OM were significantly greater in Site A than Site B (P < 0.001) ( Table 1). There was a significant effect of phosphorous treatment on soil K, Mg, P and pH (P < 0.001). The pH of P45 was generally higher than the P0 and P15 in the two sites, but the P45 at site B was significantly higher than P0 and P15 (P < 0.01). Expectedly, the P content of P45 was significantly higher than P0 and P15 at both sites (P < 0.01) ( Table 1). Site B had significantly greater WHC than Site A.
C + N addition significantly (P < 0.01) increased soil NH 4 + -N and NO 3 − -N concentrations in the two sites with the highest increase observed in P45 and P15 plots (Fig. 1). However, the NH 4 + -N and NO 3 − -N concentrations decreased rapidly in the following week except the NO 3 − -N concentrations in site A after the second application when it further increased before decreasing afterward (Fig. 1). Site B had generally significantly higher NH 4 + -N concentrations (P = 0.05) (Fig. 1).
Precipitation, WFPS and soil temperature. Daily precipitation ranged from 0.1 to 18 mm during the experiment period, with most of the rainfall occurring in the period following the second C + N addition (Fig. 2). Thus, the cumulative rainfall (60.2 mm) during the second addition was greater than the first (41.7 mm), which is considerably dry, compared to the total mean (162.5 mm) of the previous 10 years (2009-2018) of the www.nature.com/scientificreports/ same period. WFPS decreased progressively from 96.35 to 53.19% following the first addition event, but it stayed above 65.19% for the majority of the second application period (Fig. 2). Owing to a co-occurrence of fertilization events with the preceding rainfall, the addition of C solution did not cause a further increase in soil moisture content. Average soil temperature of 15 °C was recorded in the first fertilization period, which was slightly lower than the second period (18 °C) (Fig. 2).

N 2 O and CO 2 emissions.
An increase in labile C and mineral N-availability via the applications of fertilizer and glucose-acetate-methanol mixture to P0, P15, and P45 plots in the two sites resulted in increased N 2 O emissions, reaching a maximum N 2 O flux 2 days after application before decreasing after 8 days to background flux levels for both application events (Fig. 3a). The effect of P-treatment (P = 0.029) and site (P = 0.010) was significant but the interaction of the two was not significant. The emissions associated with the P0 treatment was significantly higher (P = 0.026) compared P45 treatments, indicating that low soil phosphorous enhanced N 2 O production (Fig. 3a). The cumulative N 2 O emission was significantly higher (P = 0.021) in P0 treatment than P45 treatment in both sites (Fig. 3c). The cumulative N 2 O emission in P0 was higher than in P15 treatment in the two sites. The cumulative N 2 O emission in site B (1.04 ± 0.04 g N 2 O-N m −2 ) was significantly higher (P = 0.011) than in site A (0.88 ± 0.05 g N 2 O-N m −2 ) (Fig. 3c). Although peak N 2 O flux occurred after the first C + N addition, the cumulative N 2 O emission following the second addition was significantly higher than that of the first. Multiple successive peak CO 2 emissions were observed following C + N addition in all treatments (Fig. 3b). The CO 2 emission in P45 was higher than in P0 and significantly higher than in P15 (P = 0.036) plots in site www.nature.com/scientificreports/ A whereas, in site B, the CO 2 emission in P15 was higher than in P0 plots. There was no significant difference between CO 2 emissions at P45 and P0 in site B. There were no significant differences in cumulative CO 2 emission either between sites or site × treatment interaction (Fig. 3d). However, cumulative CO 2 emission was significantly higher (P = 0.047) in the P45 and higher in P15 than in the P0 plots (Fig. 3d), being highest overall in the P45. There was no significant variation in the resultant cumulative CO 2 emission between the first and the second application events, despite generally higher cumulative emission after the first addition.

Microbial biomass and glomalin-related soil protein (GRSP). No significant interaction of site
and treatment was observed. Total GRSP varied between the two sites with Site B having significantly higher (P < 0.01) glomalin concentrations ( Table 2). Total GRSP in P0 treatments of Site A and B were significantly greater than the P15 and P45 treatments (P < 0.01) ( Table 2).
Significant treatment effect was observed in MBN (P = 0.045) and MBP (P = 0.012) following the first and second fertilization, respectively (Fig. 4a,c). Sampling time was a significant factor (P < 0.001) in determining the microbial biomasses whose levels were considerably higher in the first and second sampling after C + N additions. In the first application, MBN was significantly greater (P < 0.010) at site A for every treatment on 07/05, 09/05, and 16/05 in the first application, whereas in the second application MBN at site A was significantly greater (P < 0.010) on 14/06 for P0 and P45 and on 05/07 for P45 treatment (Fig. 4a). Significantly greater MBP at site A was observed on 09/05 and 05/07 at P0 (P = 0.019) and P45 (P = 0.045) treatments, respectively (Fig. 4c). MBC at P0 plots of site A was significantly greater at 09/05 (P = 0.034) and 05/07 (P = 0.030) (Fig. 4b).
Plant biomass. No interaction of site and treatment was observed for plant dry matter. The dry matter yield at the end of the first C + N addition (May-June) was significantly (P < 0.01) higher than the dry matter at the end of the second application (June-July) ( Table 2). There was no noticeable difference in dry matter yield between P15 and P45 plots, but the yield in these plots was significantly greater (P < 0.01) than the yield in P0 in both sites ( Table 2). Site B during the second addition had significantly higher dry matter yield than site A at every corresponding phosphorous level plot (P = 0.034).

Correlations between N 2 O and CO 2 emissions and soil and plant parameters. The daily N 2 O
emissions were significantly correlated with MBP, MBC, NH 4 + , NO 3 − , and WFPS, but were not correlated with MBN and soil temperature by Spearman's rank correlation (Table 3). Cumulative N 2 O and CO 2 emissions were significantly correlated with Glomalin content, which was related to C, K, Mg, N, and plant biomass (Table 4).  (Table 3). A significant positive association was found among PDA, C, and N with the PDA correlated neither with cumulative N 2 O nor CO 2 emissions (Table 4).

Discussion
Carbon and mineral N availability directly influence soil N 2 O and CO 2 emissions, but the interaction with different P levels have not been systematically studied, especially under field conditions. In this study, N 2 O and CO 2 emissions were quantified from two long-term P-trials following C and N addition along a soil P gradient. Our study shows that both N 2 O and CO 2 emissions increased following co-application of C and N; however, www.nature.com/scientificreports/ the magnitude of the emissions were constrained by the P level, with the highest N 2 O and CO 2 emissions associated with P-limited and P-enriched soils, respectively (Fig. 3a-d). These results indicate that P limitation or enrichment can play an essential role in determining N 2 O and CO 2 emissions in grassland ecosystems. While N fertilization, through increasing soil NO 3 − and NH 4 + concentrations, provided the substrates for nitrifiers and denitrifiers for N 2 O production (Fig. 1), the addition of C may have promoted microbial mineralization of C from these substrates or from soil organic C pools. These findings support our first hypothesis that C + N    www.nature.com/scientificreports/ addition in P-limited soil increases N 2 O production and the second hypothesis that C mineralization is increased at higher soil P levels.

Differences in N 2 O emissions at different P levels. The differences in the N 2 O emission are most likely
to be associated with induced differences in the composition, activity and/or diversity of microbial communities in relation to different P levels. Our results suggest the greater N 2 O emission in P-limited soils (Fig. 3a,c) may be associated with higher abundances of arbuscular mycorrhizal fungi (AMF), as indicated by higher levels of glomalin ( , leading to higher N 2 O emission in P-limited plots. Glomalin is regarded as a metabolite of AMF, and its concentration is largely associated with the abundance of the AMF hyphae 39,40 . In this study, greater concentrations of glomalin were detected in P0 than P15 and P45 treatments indicating that AMF are more pronounced in plots with lower soil P availability. Typically higher MBC in P0 than P15 and P45 in both sites suggest that more carbon could be immobilized by microorganisms (Fig. 4). This may also be indicative of higher abundances of arbuscular mycorrhizal fungi. The significant correlations between daily N 2 O fluxes and MBP and MBC (Table 3) and between cumulative N 2 O emissions and glomalin content (Table 4) support the argument that microbial acquisition of C and P as well as AMF abundance were related to production of N 2 O. Arbuscular mycorrhizal fungi are recognized for their greater C assimilate demand and sink strength and thus may have had a role in the differences observed in MBC 21,44,45 . Owing to the ability of AMF to acquire immobile soil P and trade P for plant growth; they form mutually beneficial relationships with their host to satiate their C demand in P-limited soils 20 . This is in line with previous evidence that showed enhanced AMF colonisation was observed at low P in the experimental plots at Site A 10,24 . Recent findings of Okiobe et al. 28 showed a strong influence of AMF presence on promoting potential N 2 O production via an increase in hyphal density and via enhanced water stable soil aggregates, indicating possible lack of N 2 O reductase in the denitrification process. However, there are earlier studies that reported contrasting results where AMF reduced N 2 O emissions as a result of reduced soil NH 4 + availability because of enhanced transport of NH 4 + by AMF to the host plant 26,27 . Our observations showed adequate availability of NH 4 + following the C + N application (Fig. 1a,b), thus we can deduce that N 2 O may not be reduced as there was surplus substrate to facilitate denitrification.
The peak N 2 O emissions (Fig. 3a) coincided with higher WFPS, which was above 80% at the time of fertilization (Fig. 2). Higher WFPS coupled with higher available C, being served as donor electron to the denitrifiers could have stimulated denitrifying microorganisms via enhanced anaerobic conditions. While this could be true for all P treatments, the varying magnitude of N 2 O emissions could have resulted partially from the indirect effect of variable heterotrophic respiration due to differences in P availability. High P has been found to impede heterotrophic respiration in grassland ecosystems 16,17 . Similarly, O'Neill et al. 29 showed higher heterotrophic respiration in the low P than high P in an incubation experiment conducted in the same experimental site utilizing sieved soils with no plant respiration component. Lowering of soil O 2 concentration as a result of heterotrophic respiration might have promoted more suitable denitrifying conditions in the P-limited soils leading to higher denitrification-derived N 2 O production.
The significant positive correlations between NH 4 + and NO 3 − and N 2 O emissions (Table 3) suggest that N 2 O fluxes depend on the amount of mineral N available in the soil. However, the high N 2 O emission observed in site B (Fig. 3a) in all corresponding treatments might be associated with the higher soil NH 4 + (Fig. 1a,b), particularly evident following the second C + N addition event when the NH 4 + concentration at site B was approximately double the amount in site A. It is subsequent to the second application where the greatest differences and the highest cumulative N 2 O emissions occurred (Fig. 3c). Optimum soil moisture conditions (Fig. 2) and sufficient availability of NH 4 + might have formed more conducive conditions for nitrification in site B. Certainly, denitrification-related N 2 O emission could also be stimulated because equally high NO 3 − concentrations (Fig. 1c,d) were detected in site B. Approximately equal correlation of N 2 O flux with NH 4 + and NO 3 − (Table 3) indicate that both nitrification and denitrification can be important pathways of N 2 O emissions in the two sites. Microbial parameters including MBN, MBC, and MBP showed significantly lower values in site B (Fig. 4) that might suggest that more immobilization of N appears to occur in site A (Fig. 4). These microbial biomass differences might also suggest disparities in the microbial community between the two sites where a subset of the microbial group could have inherently different N-transformation pathways towards regulating N 2 O production. Unexpectedly, the PDA at site A for P0, P15, and P45 was 1.7, 0.9, and 1.4 times that of site B, respectively Table 2. Dry matter (DM) yield (kg ha −1 ), glomalin-related soil protein (GRSP) (mg g −1 BSA), and potential denitrification (PDA) (ng N 2 O-N g −1 min −1 ) values for each P treatment in Site A and Site B. Letters indicate significant differences (P < 0.05) between P treatments.  . Letters indicate significant differences (P < 0.05) between P levels within the same sampling date. www.nature.com/scientificreports/ (Table 2), confirming the presence of different genetic capacity to denitrify and also suggesting higher PDA does not necessarily guarantee higher N 2 O emission in the field. Potential denitrification was related to soil C and N (Table 4), which are significantly higher in site A than site B (Table 1). Climate parameters may have a diminishing role in explaining site differences due to that the two study sites are proximate, and have been under identical management practices. WHC in site A was higher than site B (Table 1). Wang and Cai 46 observed increasing N 2 O production with increasing WHC. The soil P level and its effect on greenhouse gas quantifications are usually unaccounted for in almost all ecosystems. This is one of the few field studies demonstrating the relationship between C, N, and P, and the impact on N 2 O emissions from grassland ecosystems. Nonetheless, further studies on the long-term interaction of C, N, and P in multiple ecosystems (soil) types under natural conditions are needed to critically appraise the influence of contrasting P fertilization on N 2 O and CO 2 emissions. To expand our insight into the role of soil P in ecosystem N cycling, future studies should focus on revealing the effect of variable P content on N transformation pathways, and their linkage to microbial community and specific functional genes. Understanding the effect of soil P on N 2 O emissions may pave the way forward to an optimised use of P and N as mitigation measures that both underpins food security and reducing greenhouse gas emissions.

Effect of C + N addition on CO 2 emissions.
Unlike the N 2 O emissions, P-enriched plots, relative to the low P, showed greater CO 2 emission following C + N addition in the long-term grassland sites (Fig. 3d). These variations could be due to the enhancement of autotrophic (root) respiration caused by P availability in the P-enriched plots 16 and reduced presence of AMF in these plots. Several studies reported increased soil respiration in response to P addition 16,47,48 because of the stimulating effect of P on aboveground and belowground biomass 16,48 . Higher aboveground plant biomass was generally observed in the P-enriched plots ( Table 2) supporting the inference that net primary production and hence autotrophic respiration was higher at high P, which is supported by the positive correlation between cumulative CO 2 emission and dry matter yield (Table 4) with a proposition that above-and below-ground biomass follow an isometric pattern. Ren et al. 16 found an annually increasing trend in root biomass and concurrently increasing autotrophic respiration induced by P fertilization in a 4-year field study in an alpine grassland. In a study performed in vivo, Del-Saz et al. 49 showed a decrease of www.nature.com/scientificreports/ root respiration via alternative respiratory pathways resulting from decreasing root exudation of carboxylates such as citrate and malate as a result of AMF colonization, which is typically initiated by P-deficiency. This demonstrates the effect of P availability on respiratory pathways, hence soil respiration. Phosphorous plays an important role in the synthesis of nucleic acid and membrane, and enzymatic activations, and sufficient P could have supported specific root respiration 23 . However, aboveground plant respiration can also significantly contribute to the total ecosystem respiration in grassland ecosystems 50 . We found no relationship between CO 2 emissions and WFPSs and temperature in the two sites (Table 3). Arbuscular mycorrhizal fungi are capable of acquiring considerable amount of N and P from soil by expanding the surface area of the root system and transport these nutrients to their host plant in exchange for photosynthetically-fixed carbon 20 . The higher glomalin concentration at the P0 (Table 2) indicated higher AM-related C input into soil, which is demonstrated by the positive relationship between glomalin and soil carbon in Table 4. This contributes to macro-aggregate formation and SOM stabilisation 51 . The positive relationship between GRSP and soil carbon in this study is in line with previous findings [52][53][54] where positive contributions of glomalin to maintaining the soil carbon pool have been reported. Clemmensen et al. 52 , in their study on boreal forests, using a combined technique of pyrosequencing of DNA-barcodes and isotopes, identified root-related fungi as important regulators of ecosystem C dynamics. Zhang et al. 55 showed the regulatory power of AMF on soil respiration as AM inhibition resulted in accelerated soil respiration due to increased availability of root-exudated carbohydrate to other microbes in the rhizosphere. These findings, together with ours, suggest that AMF may influence soil C dynamics by increasing SOC recalcitrance either via aggregation or increasing decomposition resistant C species (glomalin, chitin, etc.). This argument is supported by the significant negative correlation between glomalin and cumulative CO 2 emission ( Table 4), such that long-term plant fungi partnership at P0 has increased aggregate stabilization of C via glomalin, causing reduced CO 2 production. Greater allocation of biomass to roots delivers C to the soil and the greater the depth that rooting occurs, the lower the decomposition due to low redox potential. This can lead to the conclusion that AMF-derived C contributes more to the stable soil C pool than the carbon derived from aboveground dry matter yield ( Table 2). Aboveground biomasses in P15 and P45 plots were greater than P0 (Table 2). This is obviously due to a change in C:N:P stoichiometry caused by variable phosphorous supply. In the P0 plots, where there is a limited supply of P, plants form nutritional symbiosis leading to high C-nutrient exchange with AMF, whereas in the P-rich plots such symbiotic relationship is eliminated or reduced due to the repeated N and P fertilization. Where N and P are sufficiently available, plants invest less in mycorrhizas and AMF, instead adjusting C allocation to the aboveground biomass 20,23 . Therefore, these results underscore the need to account for soil C sequestration and C fixation by plants, in addition to CO 2 fluxes, in order to assess the impact of phosphorus fertilization on C balance of grassland ecosystems and suggest mitigation options, which is achieved through evaluation of changes in SOC over an extended time and assessment of net ecosystem exchange.

Conclusions
Soil P plays an important role in determining N 2 O and CO 2 emissions under equivalent C + N conditions. This is the first field study that shows a significant effect of differing soil P levels on the two major greenhouse gases such as N 2 O and CO 2 in temperate grassland ecosystems. Higher N 2 O emission was observed in P-limited soils whereas P-enrichment enhanced CO 2 emissions in the two permanent grassland ecosystems. P fertilization can reduce N 2 O emissions derived from N-fertilization but increase CO 2 emissions.. These findings are important in informing effective management strategies to agronomic practices underpinning an optimized use of N and P as mitigation measures for both food security and reducing greenhouse gas emissions. Furthermore, our findings highlight the need for representation of P in process-based land models with its effect on the dynamics of greenhouse gases in terrestrial ecosystems. Future studies may reveal how the interaction of C and N with P affect specific N-transformation pathways, C sources of mineralization, and microbial communities and their functional traits in these ecosystems.